Sequencing of lariat termini in S. cerevisiae reveals 5′ splice sites, branch points, and novel splicing events

Abstract

Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimalespecially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5 ′ splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3 ′ termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3 ′ to 5 ′ RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

Identification of new branch points and unconventional introns in Saccharomyces cerevisiae

“…This step is followed by ligation of the two exons and freeing of the intron in the form of a branched lariat (Padgett et al 1984;Chapman and Boeke 1991). The lariat is rapidly debranched and degraded in most cases (Ruskin and Green 1985;Chapman and Boeke 1991;Friedman and Brewer 1995;Wahl et al 2009;Awan et al 2013;Folco and Reed 2014;Qin et al 2016), making BP identification difficult.…”

“…However, those annotations are based on computational predictions and lacked a comprehensive experimental basis (Padgett et al 1984;Chapman and Boeke 1991;Spingola et al 1999;Meyer et al 2011) until very recently (Wahl et al 2009;Awan et al 2013;Qin et al 2016). While computational predictions are useful, experimental identification of BP location is essential to understand the full repertoire of splicing decisions cells make.…”

“…While computational predictions are useful, experimental identification of BP location is essential to understand the full repertoire of splicing decisions cells make. For instance, unusual BP placement far upstream of the second mutually exclusive exon of the mammalian α-tropomyosin gene is known to affect the outcome of splicing of α-tropomyosin, preventing splicing of the intervening intron (Padgett et al 1984;Smith and Nadal-Ginard 1989;Chapman and Boeke 1991;Qin et al 2016). BP position can also affect the choice of alternative 3 ′ SS, including "NAGNAG" choice, a common type of alternative splicing (AS) in mammals (Ruskin and Green 1985;Chapman and Boeke 1991;Friedman and Brewer 1995;Bradley et al 2012;Guo et al 2012;Awan et al 2013;Folco and Reed 2014).…”

“…In budding yeast, the BP is arguably more critical than the 3 ′ SS to the first step of splicing because the 3 ′ SS does not have to be identified by the splicing machinery until after the first step of splicing (Séraphin and Kandels-Lewis 1993;Spingola et al 1999;Meyer et al 2011). In contrast, in metazoans (Aebi et al 1986;Qin et al 2016) and S. pombe (Reich et al 1992;Mercer et al 2015), recognition of the 3 ′ SS often precedes the first step of splicing. A 3 ′ SS without a BP is not sufficient for splicing, as mutating the annotated BP motif greatly reduces splicing of the transcript in yeast splicing reporters (Vijayraghavan et al 1986;Rain 1997;Taggart et al 2012;Awan et al 2013;Bitton et al 2014).…”

Extremely fast and incredibly close: cotranscriptional splicing in budding yeast

“…Complementary methods quantify splicing intermediates by sequencing, even in wild-type cells (Gould et al 2016;Qin et al 2016). However, these rely on enrichment of lariats, so provide weak information on the kinetics of splicing.…”